Cancer is the second leading cause of human mortality. In the United States alone, cancer causes the death of well over a half-million people annually, with some 1.4 million new cases diagnosed per year. Carcinomas of the breast, prostate, lung, colon, pancreas, and ovary are particularly lethal, because of the propensity of these cancers to produce fatal metastases. Even those cancer patients who initially survive their primary cancers can suffer physical and psychological debilitation following treatment, and many experience a recurrence of the disease.
Cancer morbidity and mortality can, however, be greatly reduced by early diagnosis and treatment. Thus, an intensive worldwide effort had been aimed at identifying truly specific, early-stage diagnostic and prognostic markers for cancers. Research efforts in these areas have increased the availability of useful molecular diagnostic and diagnostic imaging technologies, but progress in this area has been slow and generally uneven.
For example, alleles of the BRCA1 and BRCA2 genes have been linked to hereditary and early-onset breast cancer (Wooster, et al., Science, 265: 2088-2090 (1994)). Detection of mutated BRCA1 and BRCA2 alleles or their gene products has therefore been proposed as a means for detecting breast cancer (Miki, et al., supra). However, the BRCA1 and 2 genes are of limited use as a cancer markers, because mutations in these genes fail to account for the majority of breast cancers (Ford, et al., British J. Cancer, 72: 805-812 (1995)). Moreover, breast cancer has been generally classified into four different stages (I-IV), with Stage I being early cancerous and Stage IV being metastatic. Markers specific for each stage have yet to be identified.
SELDI-TOF mass spectrometry has been used for detecting putative breast cancer markers from serum, nipple aspirate fluid and others but the results were both confusing and contradictory. For example, markers reported by Laronga et al (Dis. Markers 19: 229-38 (2003)) was later found by Gast et al to be unreliable (Cancer Biomarkers 2: 235-48 (2006)). Gene profiling has been suggested as an alternative to the unreliable SELDI method. Several gene profiling kits such as MammaPrint and OncotypeDX are available for detecting breast cancer. They are based on the principle that when genes are damaged (as in the case of breast cancer), they will turn on some other genes that should normally be off and silence others that should be on. The premise is that the pattern of genes turned on in diseased tissue is abnormal, and can be used to predict disease progression, long-term survival, and how well patients will respond to drugs and radiation. Predictions based on gene profiles are not accurate and sometimes wrong.
Another cancer with a relatively high incidence and poor prognosis is pancreatic adenocarcinoma (PA). The molecular basis underlying the pathogenesis of PA is unknown, and the ability to detect early lesions for resection remains a challenge despite advances in diagnostic imaging methods. Furthermore, distinguishing PA from benign pancreatic diseases, especially chronic pancreatitis, is difficult because of the similarities in radiological and imaging features and the lack of clinical symptoms specific for PA.
Serologic assays for breast cancer and PA are easily performed, inexpensive, analytically-sensitive and can be serially performed over time with relative ease. To date, however, there exists no serologic assay which can specifically and reliably detect these or other cancers.
For example, several non-specific breast cancer markers, including glycosyl transferases (Ip et al., Cancer Res., 38: 723-728 (1978); Dao et al., J. Natl. Cancer Inst., 65: 529-534 (1980)) and glycolipids (Kloppel et al., Proc. Natl. Acad. Sci. USA, 74: 3011-3013 (1977)) can be detected by serologic assays. Serum-based immunoassays can also detect circulating human mammary epithelial antigens which may be present in elevated amounts in the plasma of breast cancer patients (Ceriani et al., Proc. Natl. Acad. Sci. USA, 79: 5420-5424 (1982); Hayes, J. Clin. Invest., 75: 1671-1678 (1985)). However, detection of these markers and antigens is not a widely accepted clinical assay for breast cancer.
Serum-based immunoassays have been used to detect blood group-related antigens and glycoprotein markers commonly used as clinical tumor markers for PA, such as CA19-9, CA72-4, CA125, and more recently CA242. However, there are contradictory reports about the specificity and sensitivity of these immunoassays. For example, the specificity of the CA19-9 serum assay for detecting PA ranged from 69% to 93%, and the sensitivity varied between 46% and 98% (Eskelinen et al, Scand. J. Gastroenterol. 34: 833-844 (1999)). CA19-9 antigen also exhibited elevated serum levels in some benign pancreatic diseases (Slesak et al., Cancer 89: 83-88 (2000)).
Furthermore, in many serologic assays, the presence of cancer markers can be obscured by major serum proteins such as serum protein (which constitutes approximately 50% of serum proteins), immunoglobulin G (IgG), heptoglobin, and alpha-1-antitrypsin.
Conventional two-dimensional polyacrylamide gel electrophoresis (“2-D PAGE”), first developed by O'Farrell (J. Biol. Chem. 250: 4007-4021, 1975), is a common serologic assay used to detect cancer markers. In this method, proteins are first separated under denaturing conditions according to their isoelectric points, followed by separating the proteins according to their molecular weights in a second dimension in the presence of an ionic detergent.
In order to carry out biological functions, proteins usually form complexes with other proteins. On average, a protein forms complexes with 4 or 5 different partners. Understanding protein-protein interaction is therefore the key to unlock the mystery of cell function, and how diseases occur and progress. The commonly used 2-D polyacrylamide gel electrophoresis system (2-D PAGE) cannot separate protein complexes because it is carried out under denaturing conditions which destroys all protein complexes.
Conventional 2-D PAGE has other disadvantages. The separation of serum proteins on the gel involves multiple steps, and generally takes one to two days to complete. The proteins must then be “blotted,” or transferred onto high protein binding capacity, low porosity polymer membranes so they can be detected by staining, immunodetection (e.g., Western blot), mass spectrometry, amino acid sequence analysis or the like. The blotting step is also time consuming, and can result in loss of separated protein due to inefficient transfer out of the gel. For example, the retention of low molecular weight proteins by nitrocellulose is influenced by the presence of methanol in the transfer buffer (Pluskal et al., Biotechniques 4: 272-283, 1986). Higher molecular weight proteins are also known to have lower transfer efficiency onto blotting membranes. Detection of small amounts of separated proteins can therefore be difficult. And as indicated above, any protein-protein interactions or biological activities of the separated proteins are not preserved under the denaturing conditions used in conventional 2-D PAGE techniques.
Conventional 2-D PAGE also typically employs aqueous buffers, because such buffers provide the high conductivity needed for protein separation. However, the use of aqueous buffers can generate excessive heat during electrophoresis, which can damage the protein sample or electrophoretic equipment. The use of aqueous buffers also prevents the efficient separation of hydrophobic proteins.
On average, a protein forms complexes with 4 or 5 different partners. The commonly used 2-D polyacrylamide gel electrophoresis system (2-D PAGE) cannot separate protein complexes from one another while maintaining the integrity of the complexes because 2-D PAGE is carried out under denaturing conditions which destroys all protein complexes. An electrophoretic system that is fast, requiring very small amount of serum sample and also allows the separation of both proteins and protein complexes, while maintaining the integrity of the latter, would be desirable for detecting cancer and other disease marker proteins.
A one dimensional electro-separation method has been developed which uses water-miscible organic solvents to separate small molecules on separation substrates such as filter paper (see U.S. Pat. No. 4,146,454; Haber N., PNAS USA, 79:272-276, 1982; and Haber N., Biotechnic & Histochemistry, 73: 59-70, 1998). In this method, which is called “electro-molecular propulsion” or “EMP,” an electronic charge is imposed on the molecules by an unknown mechanism, which causes the molecules migrate within an applied electrical field. EMP is therefore different from conventional electrophoresis systems, in which movement of molecules in an electric field depends on ionic species dissolved in an electrolytically conductive medium. See Haber N., Biotechnic & Histochemistry, 1998, supra.
The EMP technique does not appear suitable for analysis of ampholytic biopolymers such as proteins, primarily because the substrates used in the EMP process do not bind proteins well, and proteins separated by EMP begin to diffuse on the substrates almost immediately after cessation of the electric current. This diffusion of proteins has greatly limited the usefulness of the EMP process, and no 2-D protein separation procedure employing this technique has been reported.
Therefore, even with advances in molecular diagnostic and diagnostic imaging techniques, a simple and effective assay for breast cancer and PA (and indeed other cancers) remains lacking. What is needed, therefore, is a method for rapidly detecting cancer and other disease marker proteins present in serum or other bodily fluids, for example by electrophoretic separation. The electrophoretic method should employ low conductivity, organic solvent buffers compatible with hydrophilic, hydrophobic and low molecular weight proteins. The buffers should also have low conductivity so as to minimize heat generation during electrophoretic separation, and are preferably non-denaturing to preserve protein binding interactions and biological activities. Ideally, the electrophoretic separation substrate should minimize diffusion of the separated molecules after electrophoresis is completed, and eliminate the need for transferring the separated molecules from the separation matrix onto a blotting membrane.